Cells containing melanin pigmentation aid vision, protect against UV radiation, and can transform into neoplasms. This proposal is based upon three related discoveries in our lab. 1) The zebrafish hypopigmentation mutant, golden, shows a melanosomal phenotype similar to that seen in light-skinned people. 2) The affected gene in this mutant is slc24a5, which encodes a putative K-dependent-Na/Ca exchanger. 3) A coding polymorphism in the human orthologue SLC24A5 is an important determinant of the difference in human skin color between Africans and Europeans. Our long-term goal is to elucidate the role and mechanism of the SLC24A5 protein in melanogenesis. We propose a collaborative, multidisciplinary approach that includes ultrastructural analysis, functional genomics, and transport studies. We are testing the hypothesis that slc24a5 is a calcium exchanger located in the membranes of melanosomes or their precursors, and that the organellar ionic milieu established by its transport activity is important in melanosome morphogenesis and pigment formation. Specific Aim 1 seeks to determine the subcellular distribution of the slc24a5 protein among melanosomes and their precursors using double-label, confocal immunofluorescence microscopy, subcellular fractionation, and immunoelectron microscopy. Specific Aim 2 combines ultrastructural analysis with antisense morpholino knockdown to define the morphogenetic roles of slc24a5. Specific Aim 3 seeks to establish the transport properties of the slc24a5 protein using a combination of calcium imaging, electrophysiology, and Ca-45 transport studies. Specific Aim 4 addresses the functional consequences of the common human polymorphism in SLC24A5. Findings from the proposed work will enhance our understanding of melanosome morphogenesis and melanin pigmentation in vertebrates, including humans. Melanin-containing cells aid vision, protect us from UV radiation, and when transformed, become one of the deadliest cancers in humans, malignant melanoma. This work will clarify how a newly-discovered sodium- calcium exchanger protein, first characterized in zebrafish, affects pigmentation, and lays a foundation for drugs to treat skin pigmentation problems, and perhaps malignant melanoma.